scholarly journals The Length of Promoter Sequence Affects The De Novo Initiation By T7 RNA Polymerase in Vitro: New Insights Into The Evolution of Promoters For Single Subunit RNA Polymerases

2019 ◽  
Author(s):  
Ramesh Padmanabhan ◽  
Dennis Miller
Keyword(s):  
Rna Polymerase ◽  
Transcription Initiation ◽  
De Novo ◽  
Promoter Sequence ◽  
Rna Polymerases ◽  
T7 Rna Polymerase ◽  
Promoter Sequences ◽  
Promoter Recognition ◽  
Single Subunit

1.1AbstractRNA polymerases (RNAPs) differ from other polymerases in that they can bind promoter sequences and initiate de novo transcription. Promoter recognition requires the presence of specific DNA binding domains in the polymerase. The structure and mechanistic aspects of transcription by the bacteriophage T7 RNA polymerase (T7 RNAP) are well characterized. This single subunit RNAP belongs to the family of RNAPs which also includes the T3, SP6 and mitochondrial RNAPs. High specificity for its promoter, the requirement of no additional transcription factors, and high fidelity of initiation from a specific site in the promoter makes it the polymerase of choice to study the mechanistic aspects of transcription. The structure and function of the catalytic domains of this family of polymerases are highly conserved suggesting a common mechanism underlying transcription. Although the two groups of single subunit RNAPs, mitochondrial and bacteriophage, have remarkable structural conservation, they recognize quite dissimilar promoters. Specifically, the bacteriophage promoters recognize a 23 nucleotide promoter extending from −17 to + 6 nucleotides relative to the site of transcription initiation, while the well characterized promoter recognized by the yeast mitochondrial RNAP is nine nucleotides in length extending from −8 to +1 relative to the site of transcription initiation. Promoters recognized by the bacteriophage RNAPs are also well characterized with distinct functional domains involved in promoter recognition and transcription initiation. Thorough mutational studies have been conducted by altering individual base-pairs within these domains. Here we describe experiments to determine whether the prototype bacteriophage RNAP is able to recognize and initiate at truncated promoters similar to mitochondrial promoters. Using an in vitro oligonucleotide transcriptional system, we have assayed transcription initiation activity by T7 RNAP. When a complete or almost complete (20 to 16 nucleotide) double stranded T7 RNAP promoter sequence is present, small RNA’s are produced through template-independent and promoter-dependent stuttering corresponding to abortive initiation, and this effect was lost with a scrambled promoter sequence. When partial double stranded promoter sequences (10 to 12 nucleotides) are supplied, template dependent de novo initiation of RNA occurs at a site different from the canonical +1-initiation site. The site of transcription initiation is determined by a recessed 3’ end based paired to the template strand of DNA rather than relative to the partial promoter sequence. Understanding the mechanism underlying this observation helps us to understand the role of the elements in the T7 promoter, and provides insights into the promoter evolution of the single-subunit RNAPs.

scholarly journals Syn5 RNA polymerase synthesizes precise run-off RNA products

2013 ◽  
Vol 42 (5) ◽  
pp. e33-e33 ◽  
Author(s):  
Bin Zhu ◽  
Stanley Tabor ◽  
Charles C. Richardson
Keyword(s):  
Rna Polymerase ◽  
Rna Synthesis ◽  
Promoter Sequence ◽  
T7 Rna Polymerase ◽  
Bacteriophage T7 ◽  
Intermediate Products ◽  
Run Off ◽  
Rna Fragments ◽  
Single Subunit

Abstract The enzyme predominantly used for in vitro run-off RNA synthesis is bacteriophage T7 RNA polymerase. T7 RNA polymerase synthesizes, in addition to run-off products of precise length, transcripts with an additional non-base-paired nucleotide at the 3′-terminus (N + 1 product). This contaminating product is extremely difficult to remove. We recently characterized the single-subunit RNA polymerase from marine cyanophage Syn5 and identified its promoter sequence. This marine enzyme catalyses RNA synthesis over a wider range of temperature and salinity than does T7 RNA polymerase. Its processivity is >30 000 nt without significant intermediate products. The requirement for the initiating nucleotide at the promoter is less stringent for Syn5 RNA polymerase as compared to T7 RNA polymerase. A major difference is the precise run-off transcripts with homogeneous 3′-termini synthesized by Syn5 RNA polymerase. Therefore, the enzyme is advantageous for the production of RNAs that require precise 3′-termini, such as tRNAs and RNA fragments that are used for subsequent assembly.

scholarly journals Np4A alarmones function in bacteria as precursors to RNA caps

2020 ◽  
Vol 117 (7) ◽  
pp. 3560-3567 ◽  
Author(s):  
Daniel J. Luciano ◽  
Joel G. Belasco
Keyword(s):  
Rna Polymerase ◽  
Transcription Initiation ◽  
Promoter Sequence ◽  
Rna Degradation ◽  
Initiation Site ◽  
Bacterial Cells ◽  
E Coli ◽  
N Incorporation ◽  
Nascent Transcripts

Stresses that increase the cellular concentration of dinucleoside tetraphosphates (Np4Ns) have recently been shown to impact RNA degradation by inducing nucleoside tetraphosphate (Np4) capping of bacterial transcripts. However, neither the mechanism by which such caps are acquired nor the function of Np4Ns in bacteria is known. Here we report that promoter sequence changes upstream of the site of transcription initiation similarly affect both the efficiency with which Escherichia coli RNA polymerase incorporates dinucleoside polyphosphates at the 5′ end of nascent transcripts in vitro and the percentage of transcripts that are Np4-capped in E. coli, clear evidence for Np4 cap acquisition by Np4N incorporation during transcription initiation in bacterial cells. E. coli RNA polymerase initiates transcription more efficiently with Np4As than with ATP, particularly when the coding strand nucleotide that immediately precedes the initiation site is a purine. Together, these findings indicate that Np4Ns function in bacteria as precursors to Np4 caps and that RNA polymerase has evolved a predilection for synthesizing capped RNA whenever such precursors are abundant.

scholarly journals Structures of E. coli σS-transcription initiation complexes provide new insights into polymerase mechanism

2016 ◽  
Vol 113 (15) ◽  
pp. 4051-4056 ◽  
Author(s):  
Bin Liu ◽  
Yuhong Zuo ◽  
Thomas A. Steitz
Keyword(s):  
Rna Polymerase ◽  
Conformational Changes ◽  
Active Site ◽  
Transcription Initiation ◽  
De Novo ◽  
Addition Reaction ◽  
Specific Interactions ◽  
Promoter Recognition ◽  
Nucleotide Addition ◽  
Promoter Dna

In bacteria, multiple σ factors compete to associate with the RNA polymerase (RNAP) core enzyme to form a holoenzyme that is required for promoter recognition. During transcription initiation RNAP remains associated with the upstream promoter DNA via sequence-specific interactions between the σ factor and the promoter DNA while moving downstream for RNA synthesis. As RNA polymerase repetitively adds nucleotides to the 3′-end of the RNA, a pyrophosphate ion is generated after each nucleotide incorporation. It is currently unknown how the release of pyrophosphate affects transcription. Here we report the crystal structures of E. coli transcription initiation complexes (TICs) containing the stress-responsive σS factor, a de novo synthesized RNA oligonucleotide, and a complete transcription bubble (σS-TIC) at about 3.9-Å resolution. The structures show the 3D topology of the σS factor and how it recognizes the promoter DNA, including likely specific interactions with the template-strand residues of the −10 element. In addition, σS-TIC structures display a highly stressed pretranslocated initiation complex that traps a pyrophosphate at the active site that remains closed. The position of the pyrophosphate and the unusual phosphodiester linkage between the two terminal RNA residues suggest an unfinished nucleotide-addition reaction that is likely at equilibrium between nucleotide addition and pyrophosphorolysis. Although these σS-TIC crystals are enzymatically active, they are slow in nucleotide addition, as suggested by an NTP soaking experiment. Pyrophosphate release completes the nucleotide addition reaction and is associated with extensive conformational changes around the secondary channel but causes neither active site opening nor transcript translocation.

scholarly journals Identification of Drosophila Promoter Using Positional Differential Matrix and Support Vector Machine from Sequence Data

1970 ◽  
Vol 18 (2) ◽  
pp. 123-130
Author(s):  
Azizul Haque ◽  
Firoz Anwar ◽  
Taskeed Jabid ◽  
Syed Murtuza Baker ◽  
Haseena Khan ◽  
...  
Keyword(s):  
Transcription Initiation ◽  
Sequence Data ◽  
Focal Point ◽  
Living Organism ◽  
Promoter Sequence ◽  
Computational Method ◽  
Support Vector ◽  
Promoter Sequences ◽  
Promoter Recognition ◽  
Transcription Initiation Complex

Promoter region plays an important role in controlling gene expression of any living organism. It regulates gene transcription by providing space to the RNA polymerase and transcription factors to bind and interact with. Binding of appropriate transcription initiation complex is determined by the specific promoter sequence carrying gene specific motifs. The promoter recognition process is a part of the complex process where genes interact with each other over time and actually regulates the whole working process of a cell. Thus computational method for identifying promoter is a focal point for researchers. This paper presents an algorithm for identifying Drosophila melanogaster promoter using differential positional frequency matrix between promoter and non-promoter sequences which shows maximum 90.36% tenfold cross validation accuracy. The proposed method exhibits greater accuracy for detecting promoters. Also higher sensitivity and specificity results elucidate that the proposed method is less prone to false negatives and false positives compared to some other existing methods.  Key words:  Drosophila, Promoter, Sequence data D.O.I. 10.3329/ptcb.v18i2.3394 Plant Tissue Cult. & Biotech. 18(2): 123-130, 2008 (December)

scholarly journals “CapZyme-Seq” comprehensively defines promoter-sequence determinants for RNA 5’ capping with NAD+

2017 ◽  
Author(s):  
Irina O. Vvedenskaya ◽  
Jeremy G. Bird ◽  
Yuanchao Zhang ◽  
Yu Zhang ◽  
Xinfu Jiao ◽  
...  
Keyword(s):  
Transcription Initiation ◽  
High Throughput Sequencing ◽  
Promoter Sequence ◽  
Promoter Sequences ◽  
E Coli ◽  
Sequencing Method ◽  
Decapping Enzymes ◽  
General Method

SUMMARYNucleoside-containing metabolites such as NAD+ can be incorporated as “5′ caps” on RNA by serving as non-canonical initiating nucleotides (NCINs) for transcription initiation by RNA polymerase (RNAP). Here, we report “CapZyme-Seq,” a high-throughput-sequencing method that employs NCIN-decapping enzymes NudC and Rai1 to detect and quantify NCIN-capped RNA. By combining CapZyme-Seq with multiplexed transcriptomics, we determine efficiencies of NAD+ capping by Escherichia coli RNAP for ~16,000 promoter sequences. The results define preferred transcription start-site (TSS) positions for NAD+ capping and define a consensus promoter sequence for NAD+ capping: HRRASWW (TSS underlined). By applying CapZyme-Seq to E. coli total cellular RNA, we establish that sequence determinants for NCIN capping in vivo match the NAD+-capping consensus defined in vitro, and we identify and quantify NCIN-capped small RNAs. Our findings define the promoter-sequence determinants for NCIN capping with NAD+ and provide a general method for analysis of NCIN capping in vitro and in vivo.

scholarly journals N4 RNA Polymerase II, a Heterodimeric RNA Polymerase with Homology to the Single-Subunit Family of RNA Polymerases

2002 ◽  
Vol 184 (18) ◽  
pp. 4952-4961 ◽  
Author(s):  
S. H. Willis ◽  
K. M. Kazmierczak ◽  
R. H. Carter ◽  
L. B. Rothman-Denes
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Sequence Similarity ◽  
Rna Polymerases ◽  
Transcriptional Analysis ◽  
Amino Acid Residues ◽  
Genes Encoding ◽  
Single Subunit

ABSTRACT Bacteriophage N4 middle genes are transcribed by a phage-coded, heterodimeric, rifampin-resistant RNA polymerase, N4 RNA polymerase II (N4 RNAPII). Sequencing and transcriptional analysis revealed that the genes encoding the two subunits comprising N4 RNAPII are translated from a common transcript initiating at the N4 early promoter Pe3. These genes code for proteins of 269 and 404 amino acid residues with sequence similarity to the single-subunit, phage-like RNA polymerases. The genes encoding the N4 RNAPII subunits, as well as a synthetic construct encoding a fusion polypeptide, have been cloned and expressed. Both the individually expressed subunits and the fusion polypeptide reconstitute functional enzymes in vivo and in vitro.

scholarly journals Transcription from the P3 Promoter of the Bacillus subtilis spx Gene Is Induced in Response to Disulfide Stress

2006 ◽  
Vol 189 (5) ◽  
pp. 1727-1735 ◽  
Author(s):  
Montira Leelakriangsak ◽  
Peter Zuber
Keyword(s):  
Bacillus Subtilis ◽  
Rna Polymerase ◽  
Intergenic Region ◽  
Transcription Initiation ◽  
Transcriptional Start Site ◽  
Promoter Sequence ◽  
Primer Extension ◽  
Control Element ◽  
Start Site

ABSTRACT The spx gene of Bacillus subtilis encodes a global regulator that controls transcription initiation in response to oxidative stress by interaction with RNA polymerase (RNAP). It is located in a dicistronic operon with the yjbC gene. The spx gene DNA complements an spx null mutation with respect to disulfide stress resistance, suggesting that spx is transcribed from a promoter located in the intergenic region of yjbC and spx. Transcription of the yjbC-spx operon has been reported to be driven by four promoters, three (P1, P2, and PB) residing upstream of yjbC and one (PM) located in the intergenic region between yjbC and spx. Primer extension analysis uncovered a second intergenic promoter, P3, from which transcription is elevated in cells treated with the thiol-specific oxidant diamide. P3 is utilized by the σA form of RNA polymerase in vitro without the involvement of a transcriptional activator. Transcriptional induction from P3 did not require an Spx-RNAP interaction and was observed in a deletion mutant lacking DNA upstream of position −40 of the P3 promoter start site. Deletion mutants with endpoints 3′ to the P3 transcriptional start site (positions +5, +15, and +30) showed near-constitutive transcription at the induced level, indicating the presence of a negative control element downstream of the P3 promoter sequence. Point mutations characterized by bgaB fusion expression and primer extension analyses uncovered evidence for a second cis-acting site in the P3 promoter sequence itself. The data indicate that spx transcription is under negative transcriptional control that is reversed when disulfide stress is encountered.

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